Sperm motility is a tightly regulated process. One of the crucial factors determining the swimming of the sea-urchin sperm is an elevation of intracellular pH (pH(i)). The possibility that its hyperpolarisation-activated cyclic nucleotide-gated channel (SpHCN) is modulated directly by pH is addressed here. Site-directed mutagenesis showed that histidine 518 from the linker connecting the S6 helix with the cyclic nucleotide binding domain is responsible for the pH modulation of current kinetics and voltage dependence of activation. The effect of mutating histidine 518 to serine (H518S) on the time constant of activation was maximal at pH 6.4: 180+/-20 ms in the wild-type (wt) but only 56+/-10 ms in the H518S mutant channel. Furthermore, histidine 518 accounted for 31% of the shift in the voltage of half activation ( V(1/2)) in wt following a pH change from 6.4 to 8.4. The mutation H518S also shifted V(1/2) by 19 mV at pH 7.4 (-50.2+/-0.2 and -69+/-2 mV for H518S and wt, respectively). This indicates that histidine 518 couples voltage sensing to gating. The wt and H518S channels had a different affinity for cyclic adenosine monophosphate (cAMP) (IC(50) 1.0+/-0.02 and 2.5+/-0.06 microM, respectively). Changes in pH(i) also modulated channel selectivity.